Combination of syrosingopine and mitochondrial inhibitors for the treatment of cancer and immunosuppression

ABSTRACT

The invention relates to a combination of syrosingopine and a mitochondrial inhibitor, e.g. metformin or oligomycin, and the use of the combination of syrosingopine and a mitochondrial inhibitor for the treatment of cancer and for achieving immunosuppression. The invention also relates to a fluorescence-based method for predicting syrosingopine sensitivity of a cancer cell.

FIELD OF THE INVENTION

The invention relates to a combination of syrosingopine and amitochondrial inhibitor, e.g. metformin or oligomycin, and the use ofthe combination of syrosingopine and a mitochondrial inhibitor for thetreatment of cancer and for achieving clinical immunosuppression.

BACKGROUND ART

Anti-cancer therapy utilizes a combination of therapeutic interventionssuch as surgery, radiation therapy and chemotherapy. Surgery andradiation therapy are generally confined locally to the main site oftumor growth, while chemotherapy is applied to prevent tumor re-growthor against distant tumor foci. Chemotherapeutic agents are also used toreduce tumor growth to manage disease progression when radiotherapy orsurgery is not an option.

Immunosuppressive agents are clinically used to suppress a pathologicalimmune reaction which targets the own body (autoimmunity) orovershooting immune reactions as seen in allergy. They are also used totreat transplant rejection caused by the immune system. Basic to immuneresponses is activation and proliferation of T cells following antigenicstimulation, which act in turn as helper cells for B cells, regulatorycells or effector cells. Immunosuppressive agents such as rapamycin orcyclosporine A act by inhibiting early T cell activation/proliferation.As both cancer and immune responses involve cell proliferation, someagents, for example rapamycin or its analogs, were initially used forimmunosuppression but found later application as anticancer agents(Recher et al., Blood 2005, 105:2527-34).

Chemotherapeutic drugs are most effectively used in combination therapy.The rationale is to apply drugs that work via different mechanisms inorder to decrease the probability of developing drug-resistant cancercells. Combination therapy also allows, for certain drug combinations,an optimal combined dose to minimize side effects. This is crucial asstandard chemotherapeutic agents target essential cellular process suchas DNA replication, cell division or induce DNA damage and thus have ageneral cytotoxic effect. Finally, combination treatment of twocompounds may uncover unanticipated synergisms and trigger effects notinduced by a single compound. In recent years, drugs are also used in aneoadjuvant setting, i.e. prior to surgery, to reduce the tumor mass orto improve long-term survival.

Syrosingopine is a synthetic derivative of reserpine, ananti-hypertensive and anti-psychotic agent (J.A.M.A., Vol. 170, Nr. 17,Aug. 22, 1959, p. 2092). Syrosingopine was introduced clinically in thelate 1950s. The reserpines are rarely used today due to the developmentof better drugs with fewer side-effects. Reserpine acts by inhibition ofthe vesicular monoamine transporter leading to catecholamine depletionand this mode of action is believed to be shared by all the reserpinederivatives with an anti-hypertensive effect. Although it has beenclinically used, syrosingopine is relatively poorly studied compared toreserpine and has never been investigated as an anti-cancer agent.

Mitochondria contain the energy generating system of a cell, wherebyelectrons from metabolism pass through complexes I-IV of the electrontransfer chain (ETC) leading to extrusion of protons from complex I, Illand IV and to a reflux of protons through complex V with concomitantformation of chemical energy in the form of adenosine triphosphate(ATP). Oxygen serves as the ultimate electron acceptor and is reduced toH₂O. Critical in this process is the inner mitochondrial membrane, asprotons extruded from the complexes pass from the matrix through thismembrane into the inter-membrane space, generating a positive membranepotential of 150-200 mV. Dyes such as TMRM (tetramethylrhodamine methylester) pass this membrane and accumulate in the mitochondrial matrix,whereby the intensity of the fluorescent signal depends on the strengthof the membrane potential. A number of well described agents inhibitmitochondrial function and may be regarded as mitochondriotoxic agents.So called uncoupling agents such as FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) uncouple the flow of protonsfrom ATP synthesis, leading to a collapse of the membrane potential withresulting loss of ATP synthesis. A number of well describedmitochondrial inhibitors target the different complexes of the ETCincluding metformin, rotenone, epiberberine, piericidin A (allinhibitors of complex I), sodium malonate and thenoyltrifluoroacetone(inhibitors of complex II), antimycin A (complex III inhibitor),potassium cyanide and sodium azide (inhibitors of complex IV), andoligomycin (complex V inhibitor). Mitochondria are believed to beancestrally engulfed bacteria. They contain a DNA genome encodingseveral components of the ETC, as well as components of themitochondrial ribosome. Agents targeting the mitochondrial genome suchas certain HIV-inhibitors of the class of nucleoside analogs, e.g.stavudine (D4T), are toxic for mitochondria as they ultimately destroythe ETC and the mitochondrial energy generating system.

Metformin is a widely used biguanide drug for type 2 diabetes. It isrelated to buformin and phenformin, two biguanides not used anymore indiabetes due to toxicity. Metformin is safe and well-tolerated and hasbeen used in long-term management of diabetes for over 50 years and isthe most-prescribed anti-diabetic drug worldwide. The main clinicalbenefit of metformin in the treatment of type 2 diabetes is thesuppression of hepatic gluco-neogenesis to reduce hyperglycemia andimproved insulin sensitivity; these effects are believed to be exertedby metformin-dependent stimulation of AMP-activated protein kinase(AMPK) activity. Basic to this effect is the fact, that metformin andother biguanides inhibit complex I of the respiratory chain (electrontransfer chain) of mitochondria (El-Mir et al., J Biol Chem 2000,275:223-228) A meta-analysis of diabetic patients receiving metforminversus an unrelated anti-diabetic agent revealed that the metforminreceiving cohort had lower incidence of cancer (Evans et al., BMJ 2005,330:1304-5; Bowker et al., Diabetes Care 2006, 29:254-8). This hasstimulated recent research into the use of metformin as an anti-canceragent or prophylactic with numerous studies and trials in progress, seeGonzalez-Angulo et al., Clin Cancer Res 2010, 16:1695-700.

SUMMARY OF THE INVENTION

The invention relates to a combination of syrosingopine and amitochondrial inhibitor, e.g. metformin (and related biguanidesphenformin and buformin) or oligomycin, and to pharmaceuticalcompositions comprising syrosingopine and a mitochondrial inhibitor.

The invention also relates to the use of a combination of syrosingopineand a mitochondrial inhibitor, e.g. metformin (and related biguanidesphenformin and buformin) or oligomycin, and of pharmaceuticalcompositions comprising syrosingopine and a mitochondrial inhibitor forthe treatment of cancer, in particular for the treatment of carcinoma,leukemia, myeloma, and lymphoma, and for achieving immunosuppression inautoimmunity, transplantation medicine and in other cases whereimmunosuppression is desirable, such as diseases of the skin, inparticular psoriasis, nervous system, in particular multiple sclerosis,and of the haemopoietic system, in particular anemias; to the use of acombination of syrosingopine and a mitochondrial inhibitor, e.g.metformin (and related biguanides phenformin and buformin) oroligomycin, for the preparation of a pharmaceutical composition for thetreatment of cancer and achieving immunosuppression, and to methods oftreatment of cancer and of achieving immunosuppression using acombination of syrosingopine and a mitochondrial inhibitor, e.g.metformin (and related biguanides phenformin and buformin) oroligomycin, or of pharmaceutical compositions comprising syrosingopineand a mitochondrial inhibitor.

Furthermore the invention relates to a method for the determinationwhether a cancerous cell is responsive to syrosingopine combinationtreatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Metformin and syrosingopine synergize to kill tumor cells invitro.

A to H: different cell lines. M=metformin, S=syrosingopine

AlamarBlue conversion cell proliferation assay. Cells were seeded into96-well microtiter plates and compounds added at the indicatedconcentration. Seeding cell density ranged from 5,000 to 15,000 cellsper well and was determined empirically for each cell type. Plates wereincubated for 3 days and proliferation determined by AlamarBlueconversion. Growth was normalized to the untreated controls and shown aspercentages (Y-axis). Panels A-E show inhibition curves in a panel ofsensitive cells, while panel F shows the growth curve in non-respondingHT1080 fibrosarcoma cells. Panels G-H show results from twonon-cancerous normal human fibroblast lines (Fib3 and Fib4). For eachcell line the left panel shows a titration of metformin with a dashedvertical line indicating the concentration (5 mM) selected forco-treatment with syrosingopine. Right panel shows a similar growthcurve with syrosingopine alone (solid line), and treatment withsyrosingopine in the presence of 5 mM metformin (dashed line). All datapoints were performed in triplicate.

FIG. 2: Time-course for long term metformin and syrosingopineco-treatment.

A549 and OPM2 cells were seeded at an initial cell density of 10,000cells/ml and 100,000 cells/ml, respectively, and compounds (M=metformin,S=syrosingopine) added at the indicated concentrations. For control,cells were incubated with solvent (0.1% DMSO). The wells were sampled atthe indicated time points for cell counting (d=days of treatment). Celldensity was plotted on the Y-axis (×1000 cells/ml).

FIG. 3: Induction of apoptosis by metformin and syrosingopineco-treatment.

OPM2 and RPM18226 cells were seeded at a density of 100,000 cells/5 mlmedium with addition of compounds. OPM2: 5 μM syrosingopine (S), 2 mMmetformin (M). RPM18226: 2 μM syrosingopine (S), 1 mM metformin (M). Nocompound=C (control), combination of syrosingopine and metformin=S+M.500 μl of culture was harvested after 3 days and the cells were washedand stained with propidium iodide/annexin V for FACS (fluorescentactivated cell sorter) analysis. Annexin V is an apoptotic marker andwas detected using a FITC-coupled antibody on the FL1-channel. Propidiumiodide (PI) exclusion staining for vital cells was detectedsimultaneously on the FL3-channel. For each measurement, the PI andannexin V negative cells represent the viable cell population (whitebars). Early apoptotic cells are PI-negative, annexin V-positive(hatched bars) and late apoptotic cells are PI-positive, annexinV-positive (black bars). Bars are plotted on the Y-axis as a percentageof the total cell population.

FIG. 4: The structurally related compound reserpine does not synergizewith metformin.

6.5 and OPM2 cells were co-treated with reserpine (R) and metformin (M)over a similar concentration range in parallel with syrosingopine (S).Left panels show the effect on growth proliferation with reserpinealone. Right panels show the growth inhibition of syrosingopine (solidline) or reserpine (dashed line) in the presence of metformin (5 mM).All data points were performed in triplicate. Growth was normalized tothe respective untreated controls and expressed as percentages (Y-axis).

FIG. 5: In vivo effect of syrosingopine and metformin co-treatment in amouse syngeneic tumor model.

6.5 cells (Colombi et al., Oncogene 2011, 30:1551-65). were injectedinto the flanks of immunocompatible DBA mice. Drug treatment commencedwhen tumors reached 100 mm² in size. Mice were separated into treatmentgroups and injected intra-peritoneally with PBS (horizontal bars),syrosingopine (S, filled diamonds, 2 mg/kg body weight), metformin (M,filled triangles, 250 mg/kg body weight) and metformin plussyrosingopine (S+M, crossed squares) daily for 15 days. Mice weresacrificed when tumor size became excessive (day 15) and tumors weredissected for measurement. (A) Chart showing tumor area (in mm²)measured over the course of the treatment, d=days. (B) Mean body weight(g) of the mice at the time of sacrifice. Figures (n) above each barindicate the number of mice per treatment group.

FIG. 6: Syrosingopine synergizes with the biguanide phenformin.

Left panel: 6.5 cells treated with the metformin analogue phenformin (P)for 3 days and growth determined by cell proliferation assay. Rightpanel: 6.5 cells co-treated with syrosingopine (S) with increasingconcentrations of phenformin. Y-axis: % growth relative to untreatedcontrols.

FIG. 7: Co-treatment of metformin and syrosingopine inhibitphytohaemagglutinin (PHA)-stimulated T cell proliferation measured atday 3.

Human peripheral blood leukocytes were cultured in presence or absenceof phytohaemagglutinin. In the presence of phytohaemagglutinin (rightpanel) the combination of increasing amounts of syrosingopine with 4 mMmetformin inhibits sharply T cell proliferation at low concentrations ofsyrosingopine (dashed line), but not in the absence of metformin(straight line). In the absence of PHA stimulation (left panel), cellssurvive (as seen microscopically) but do not proliferate at day 3. Thissurvival is only minimally affected by combining syrosingopine withmetformin (dashed line).

FIG. 8: Syrosingopine synergizes with various inhibitors ofmitochondrial function.

Murine 6.5 cells were titrated with syrosingopine (S, solid lines) aloneor in the presence of various inhibitors of mitochondrial function(dashed lines): (A) Rotenone 50 nM, (B) Piericidin A 1.25 nM, (C)Epiberberine 0.625 μM, (D) 2-Thenoyltrifluoroacetone (TTFA) 100 μM, (E)Sodium malonate 30 mM, (F) Antimycin A 5 nM, (G) KCN 5 mM, (H) Sodiumazide 1.25 mM, (I) Oligomycin 1 nM, (J) Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) 10 μM, (K) Stavudine200 μM. Cells were grown in the presence of the various compounds andgrowth inhibition measured by a proliferation assay after 3 days oftreatment. Each data point was performed in triplicate and growth wasnormalized to untreated controls set at 100%.

FIG. 9: Inhibitors of mitochondrial function alone do not kill 6.5cells.

Effect of the various mitochondrial targeting agents on growth of 6.5cells at the concentrations employed for synergistic killing withsyrosingopine. Growth is normalized to untreated cells (black bar) setat 100%. Each data point was performed in triplicate and growth wasmeasured after 3 days with a proliferation assay. (A) Rotenone, (B)Piericidin A, (C) Epiberberine, (D) 2-Thenoyltrifluoroacetone, (E)Sodium malonate, (F) Antimycin A, (G) KCN, (H) Sodium azide, (I)Oligomycin, (J) Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone(FCCP), (K) Stavudine.

FIG. 10: Synergistic killing of human cancer cells by syrosingopine andoligomycin at subnanomolar concentration.

A cell growth proliferation assay (3 days) was performed as described inFIG. 1 for (A) mouse cell line 6.5, (B) human promyelocytic leukemiacell line HL60, and (C) human multiple myeloma cell line OPM2.Syrosingopine (S) was titrated in the absence of oligomycin (solidline), in the presence of 500 pM oligomycin (dotted line) and 1 nMoligomycin (dashed line).

FIG. 11: Syrosingopine treatment increases the mitochondrial membranepotential in a dose-dependent fashion.

Murine 6.5 cells were treated with TMRM (panels 1-6), and in additionpre-incubated with FCCP (panel 2), syrosingopine at 0.1 μM (panel 3),0.5 μM (panel 4), 2 μM (panel 5) or at 5 μM (panel 6) and fluorescenceintensity was measured. Thin lines: fluorescence intensity of controlcells or FCCP-treated cells (panel 2). Heavy lines indicate cellspre-incubated additionally with syrosingopine (panels 3-6). X-axisindicates fluorescence intensity, Y-axis indicates relative cellnumbers. Left shift of the fluorescence peak after FCCP treatment (panel2) indicates reduction or loss of membrane potential, right shift(panels 4-6) indicates increase of membrane potential upon syrosingopinetreatment (compare heavy and thin lines).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a combination of syrosingopine and amitochondrial inhibitor, e.g. metformin or oligomycin, and topharmaceutical compositions comprising syrosingopine and a mitochondrialinhibitor.

The invention relates furthermore to the use of a combination ofsyrosingopine and a mitochondrial inhibitor, and of pharmaceuticalcompositions comprising syrosingopine and a mitochondrial inhibitor forthe treatment of cancer, in particular for the treatment of carcinoma,leukemia, myeloma, and lymphoma, and for the treatment of immunologicaldisorders such as autoimmunity.

“Mitochondrial inhibitors” as understood in the present inventioncomprise compounds which reduce mitochondrial activity and demonstratevarying degrees of mitochondriotoxic properties. Mitochondrialinhibitors comprise so-called uncoupling agents, which uncouple the flowof protons from ATP synthesis in mitochondria, and inhibitors thattarget different complexes of the electron transfer chain (ETC) inmitochondria, e.g. complex I, complex II, complex III, complex IV, andcomplex V of the electron transfer chain. Further compounds consideredto be mitochondrial inhibitors according to the invention aremitochondriotoxic compounds targeting the mitochondrial genome.

Many widely prescribed drugs exert side effects which are due tomitochondriotoxicity. These mitochondriotoxic drugs are also consideredmitochondrial inhibitors according to the invention. Suchmitochondriotoxic or mitochondrial inhibitory drugs synergize withsyrosingopine and represent anti-cancer agents when combined withsyrosingopine. Mitochondriotoxic drugs have been used for treatment ofvery different clinical conditions (Cohen et al., Dev Disabil Res Rev2010, 16:189-199).

Mitochondrial inhibitors according to the invention comprise:

drugs used in liver or gallbladder disease with mitochondrial sideeffects, such as tetracycline, ibuprofen, amiodarone, pirprofen,tamoxifen, valproate, chloroquine, quinidine, chlorpromazine,ketoconazole, cyclosporine A, rifampicine, and glyburine; inhibitors ofelectron transport chain complex I, such as amytal, capsaicin,haloperidol, risperidone, metformin, buformin, phenformin, bupivacaine,lidocaine, halothane, dantrolene, phenytoin, clofibrate, andfenofibrate;

inhibitors of electron transport chain complex II, such ascyclophosphamide and ketoconazole;

inhibitors of electron transport chain complex III, such as antimycin A,acetaminophen, isoflurane, and sevoflurane;

inhibitors of electron transport chain complex IV, such ascephaloridine, cefazolin, and cefalotin;

inhibitors of mitochondrial DNA synthesis, such as AZT (itovudidine),d4T (stavudine), ddl (didanosine), and ddC (zalcitabine);

uncouplers of oxidative phosphorylation, such as pentamidine,indomethacin, fluoxetine, propofol, aspirin, bubivacaine, tolcapone, anddinitrophenol;

agents which reduce molecular oxygen to superoxide via a redoxmechanism, such as doxorubicin, isoniazid, gentamycin, andfluoroquinolone; and

inhibitors of mitochondrial gene transcription, such as interferon-alphaand interferon-gamma.

Metformin is 3-(diaminomethylidene)-1,1-dimethylguanidine hydrochlorideof formula (1)

Other biguanides considered are, for example, phenformin or buformin,preferably phenformin.

Phenformin is 1-(diaminomethylidene)-2-(2-phenylethyl)guanidine offormula (2)

Syrosingopine is a derivative of reserpine of formula (3),

wherein the 4-methoxy group of the 3,4,5-trimethoxybenzoate part ofreserpine is replaced by a 4-ethoxycarbonyloxy group, as shown informula (4).

Further mitochondrial inhibitors shown to synergize with syrosingopineare:

(5A) Rotenone (Inhibitor of Mitochondrial Electron Transport ChainComplex I)

(5B) Piericidin A (Complex I Inhibitor)

(5C) Epiberberine (Complex I Inhibitor)

(5D) 2-Thenoyltrifluoroacetone (Complex II Inhibitor)

(5E) Sodium Malonate (Complex II Inhibitor): CH₂(COONa)₂ (5F) AntimycinA (Complex III Inhibitor)

(5G) Potassium Cyanide (Complex IV Inhibitor): KCN (5H) Sodium Azide(Complex IV Inhibitor): NaN₃ (51) Oligomycin (Complex V Inhibitor)

(5J) Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)(Mitochondrial Membrane Uncoupling Agent)

and

(5K) Stavudine (Mitochondrial Genotoxic Agent)

In view of the close relationship between basic compounds and their acidaddition salts, metformin, phenformin and other mitochondrial inhibitorshaving a basic nitrogen atom mean the free base or any acid additionsalt thereof. Sodium malonate, potassium cyanide and sodium azide areequivalent to other alkali salts, e.g. potassium malonate, sodiumcyanide, potassium azide, or also ammonium salts thereof. The kationiccompound epiberberine (5C) may carry any pharmaceutically acceptableanion, e.g. chloride, hydrogensulfate, methanesulfonate ordihydrogenphosphate.

Likewise, syrosingopine means the free base or any acid addition saltthereof. Salts are especially the pharmaceutically acceptable salts ofsyrosingopine.

Such salts are formed, for example, as acid addition salts, preferablywith organic or inorganic acids. Suitable inorganic acids are, forexample, halogen acids, such as hydrochloric acid, sulfuric acid, orphosphoric acid. Suitable organic acids are, for example, carboxylic,phosphonic, sulfonic or sulfamic acids, for example acetic acid,propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolicacid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelicacid, suberic acid, azelaic acid, malic acid, tartaric acid, citricacid, amino acids, such as glutamic acid or aspartic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid,adamantane carboxylic acid, benzoic acid, salicylic acid,4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid,cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonicacid, ethane-1,2-disulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid, 1,5-naphthalene-disulfonic acid, 2-, 3- or4-methyl-benzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid,dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- orN-propyl-sulfamic acid, or other organic protonic acids, such asascorbic acid.

Pharmaceutical compositions according to the invention are, for example,compositions for enteral administration, such as nasal, buccal, rectalor, especially, oral administration, and for parenteral administration,such as intravenous, intramuscular or subcutaneous administration. Thecompositions comprise syrosingopine and a mitochondrial inhibitor, e.g.metformin or oligomycin, alone or, preferably, together with apharmaceutically acceptable carrier. The dosage of the combination ofsyrosingopine and the mitochondrial inhibitor depends upon the diseaseto be treated and upon the species, its age, weight, and individualcondition, the individual pharmacokinetic data, and the mode ofadministration.

The pharmaceutical compositions comprise from approximately 1% toapproximately 95% of the combination of syrosingopine and amitochondrial inhibitor, e.g. metformin or oligomycin, single-doseadministration forms comprising in the preferred embodiment fromapproximately 20% to approximately 90% combination of syrosingopine anda mitochondrial inhibitor, and forms that are not of single-dose typecomprising in the preferred embodiment from approximately 5% toapproximately 20% combination of syrosingopine and mitochondrialinhibitor. Unit dose forms are, for example, coated and uncoatedtablets, ampoules, vials, suppositories, or capsules. Further dosageforms are, for example, ointments, creams, pastes, foams, tinctures,drops, sprays, and dispersions. Examples are capsules containing fromabout 0.05 g to about 1.0 g combination of syrosingopine andmitochondrial inhibitor.

The pharmaceutical compositions of the present invention are prepared ina manner known per se, for example by means of conventional mixing,granulating, coating, dissolving or lyophilizing processes.

Preference is given to the use of solutions of the combination ofsyrosingopine and a mitochondrial inhibitor, e.g. metformin oroligomycin, and also suspensions or dispersions, especially isotonicaqueous solutions, dispersions or suspensions which, for example in thecase of lyophilized compositions comprising the combination ofsyrosingopine and a mitochondrial inhibitor, alone or together with acarrier, for example mannitol, can be made up before use. Thepharmaceutical compositions may be sterilized and/or may compriseexcipients, for example preservatives, stabilizers, wetting agentsand/or emulsifiers, solubilizers, salts for regulating osmotic pressureand/or buffers and are prepared in a manner known per se, for example bymeans of conventional dissolving and lyophilizing processes. The saidsolutions or suspensions may comprise viscosity-increasing agents,typically sodium carboxymethylcellulose, carboxymethylcellulose,dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g.Tween 80® (polyoxyethylene(20)sorbitan mono-oleate).

Suspensions in oil comprise as the oil component the vegetable,synthetic, or semi-synthetic oils customary for injection purposes. Inrespect of such, special mention may be made of liquid fatty acid estersthat contain as the acid component a long-chained fatty acid having from8 to 22, especially from 12 to 22, carbon atoms. The alcohol componentof these fatty acid esters has a maximum of 6 carbon atoms and is amonovalent or polyvalent, for example a mono-, di- or trivalent,alcohol, especially glycol and glycerol. As mixtures of fatty acidesters, vegetable oils such as cottonseed oil, almond oil, olive oil,castor oil, sesame oil, soybean oil and groundnut oil are especiallyuseful.

The manufacture of injectable preparations is usually carried out understerile conditions, as is the filling, for example, into ampoules orvials, and the sealing of the containers.

Suitable carriers for preferred solid oral dosage forms are especiallyfillers, such as sugars, for example lactose, saccharose, mannitol orsorbitol, cellulose preparations, and/or calcium phosphates, for exampletricalcium phosphate or calcium hydrogen phosphate, and also binders,such as starches, for example corn, wheat, rice or potato starch,methylcellulose, hydroxypropyl methylcellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired,disintegrators, such as the above-mentioned starches, also carboxymethylstarch, crosslinked polyvinylpyrrolidone, alginic acid or a saltthereof, such as sodium alginate. Additional excipients are especiallyflow conditioners and lubricants, for example silicic acid, talc,stearic acid or salts thereof, such as magnesium or calcium stearate,and/or polyethylene glycol, or derivatives thereof.

Tablet cores can be provided with suitable, optionally enteric, coatingsthrough the use of, inter alia, concentrated sugar solutions which maycomprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycoland/or titanium dioxide, or coating solutions in suitable organicsolvents or solvent mixtures, or, for the preparation of entericcoatings, solutions of suitable cellulose preparations, such asacetylcellulose phthalate or hydroxypropylmethylcellulose phthalate.Dyes or pigments may be added to the tablets or tablet coatings, forexample for identification purposes or to indicate different doses ofthe combination of syrosingopine and mitochondrial inhibitor.

Pharmaceutical compositions for oral administration also include hardcapsules consisting of gelatin, and also soft, sealed capsulesconsisting of gelatin and a plasticizer, such as glycerol or sorbitol.The hard capsules may contain the combination of syrosingopine andmitochondrial inhibitor in the form of granules, for example inadmixture with fillers, such as corn starch, binders, and/or glidants,such as talc or magnesium stearate, and optionally stabilizers. In softcapsules, the combination of syrosingopine and mitochondrial inhibitoris preferably dissolved or suspended in suitable liquid excipients, suchas fatty oils, paraffin oil or liquid polyethylene glycols or fatty acidesters of ethylene or propylene glycol, to which stabilizers anddetergents, for example of the polyoxyethylene sorbitan fatty acid estertype, may also be added.

Pharmaceutical compositions suitable for rectal administration are, forexample, suppositories that consist of a combination of syrosingopineand a mitochondrial inhibitor, e.g. metformin or oligomycin, and asuppository base. Suitable suppository bases are, for example, naturalor synthetic triglycerides, paraffin hydrocarbons, polyethylene glycolsor higher alkanols.

For parenteral administration, aqueous solutions of a combination ofsyrosingopine and a mitochondrial inhibitor, or aqueous injectionsuspensions that contain viscosity-increasing substances, for examplesodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired,stabilizers, are especially suitable. The combination of syrosingopineand mitochondrial inhibitor, optionally together with excipients, canalso be in the form of a lyophilizate and can be made into a solutionbefore parenteral administration by the addition of suitable solvents.Solutions such as are used, for example, for parenteral administrationcan also be employed as infusion solutions.

Preferred preservatives are, for example, antioxidants, such as ascorbicacid, or microbicides, such as sorbic acid or benzoic acid.

On the basis of the studies described in more detail below, thecombination of a mitochondrial inhibitor, e.g. metformin of formula (1),phenformin of formula (2), or the further mitochondrial inhibitors offormula (5), in particular oligomycin of formula (51), and syrosingopineof formula (4), and pharmaceutical compositions comprising amitochondrial inhibitor and syrosingopine according to the inventionshow therapeutic efficacy against different types of cancer includingcarcinomas, sarcomas, gliomas, leukemias, lymphomas, e.g. epithelialneoplasms, squamous cell neoplasms, basal cell neoplasms, transitionalcell papillomas and carcinomas, adenomas and adenocarcinomas, adnexaland skin appendage neoplasms, mucoepidermoid neoplasms, cysticneoplasms, mucinous and serous neoplasms, ductal-, lobular and medullaryneoplasms, acinar cell neoplasms, complex epithelial neoplasms,specialized gonadal neoplasms, paragangliomas and glomus tumors, naeviand melanomas, soft tissue tumors including sarcomas, fibromatousneoplasms, myxomatous neoplasms, lipomatous neoplasms, myomatousneoplasms, complex mixed and stromal neoplasms, fibroepithelialneoplasms, synovial like neoplasms, mesothelial neoplasms, germ cellneoplasms, trophoblastic neoplasms, mesonephromas, blood vessel tumors,lymphatic vessel tumors, osseous and chondromatous neoplasms, giant celltumors, miscellaneous bone tumors, gliomas, glioblastomas,oligodendrogliomas, neuroepitheliomatous neoplasms, meningiomas, nervesheath tumors, granular cell tumors and alveolar soft part sarcomas,Hodgkin's and non-Hodgkin's lymphomas, other lymphoreticular neoplasms,plasma cell tumors, mast cell tumors, immunoproliferative diseases,leukemias including acute and chronic leukemias, miscellaneousmyeloproliferative disorders, lymphoproliferative disorders andmyelodysplastic syndromes.

On the basis of the studies described in more detail below, thecombination of a mitochondrial inhibitor, e.g. metformin of formula (1),phenformin of formula (2), or the further mitochondrial inhibitors offormula (5), in particular oligomycin of formula (5I), and syrosingopineof formula (4), and pharmaceutical compositions comprising amitochondrial inhibitor and syrosingopine according to the inventionshow therapeutic efficacy against immunological diseases sensitive toblockade of T cell proliferation including connective tissue diseasessuch as lupus erythematodes, sclerodermia, polymyositis/dermatomyositis,mixed connective tissue disease, rheumatoid arthritis, Sjögren-syndrome,panarteriitis nodosa, Wegeners granulomatosis; systemic autoimmunediseases such as rheumatoid arthritis, Goodpasture's syndrome, Wegener'sgranulomatosis, polymyalgia rheumatica, Guillain-Barré syndrome,multiple sclerosis; localized autoimmune diseases such as type 1diabetes mellitus, Hashimoto's thyroiditis, Graves' disease, celiacdisease, Crohn's disease, ulcerative colitis, Addison's disease, primarybiliary cirrhosis, autoimmune hepatitis, and giant cell arteritis.

The combination of a mitochondrial inhibitor and syrosingopine, andpharmaceutical compositions comprising syrosingopine and a mitochondrialinhibitor, e.g. metformin or oligomycin, according to the invention maybe applied in the form of fixed combinations. Such fixed combination maycontain syrosingopine and a mitochondrial inhibitor, e.g. metformin, ina relative amount (weight per weight) of between 1 to 10 and 1 to 1′000,preferably between 1 to 100 and 1 to 200, such as a combination of 1 to130, whereby the maximum recommended daily dose of metformin based onthe experience with diabetes type 2 is 2′550 mg. A fixed combination ofsyrosingopine and oligomycin may contain syrosingopine and oligomycin ina relative amount (weight per weight) of between 1′000 to 1 and 10′000to 1. Alternatively, a covalent linkage between syrosingopine and someof the mitochondrial inhibitors, e.g. metformin, may be envisaged. Forpractical reasons, the salts sodium malonate (5E), potassium cyanide(5G) and sodium azide (5H), although shown to synergize withsyrosingopine in cell tests, are not considered combination partners forsyrosingopine as anti-cancer and immunosuppressive agents.

Alternatively, the combination of syrosingopine and a mitochondrialinhibitor, e.g. metformin or oligomycin, may be applied in twodifferent, separate pharmaceutical compositions, optionally beingprovided together in a kit. The administration of syrosingopine and amitochondrial inhibitor, e.g. metformin or oligomycin, may also bestaggered, or the compounds may be given independently of one anotherwithin a reasonable time window.

Pharmaceutical compositions comprising syrosingopine and a mitochondrialinhibitor, e.g. metformin or oligomycin, may be further combined withother chemotherapeutic agents.

Therapeutic agents for possible combination are especially one or morecytostatic or cytotoxic compounds, for example a chemotherapeutic agentor several selected from the group comprising indarubicin, cytarabine,interferon, hydroxyurea, bisulfan, or an inhibitor of polyaminebiosynthesis, an inhibitor of the mTOR pathway, an inhibitor ofmTOR-complex 1 or mTOR complex 2, an inhibitor of protein kinase,especially of serine/threonine protein kinase, such as protein kinase C,or of tyrosine protein kinase, such as epidermal growth factor receptortyrosine kinase, a cytokine, a negative growth regulator, such as TGF-βor IFN-β, an aromatase inhibitor, a classical cytostatic, an inhibitorof the interaction of an SH2 domain with a phosphorylated protein, aninhibitor of Bcl-2 and modulators of the Bcl-2 family members such asBax, Bid, Bad, Bim, Nip3 and BH3-only proteins.

The combination of syrosingopine and mitochondrial inhibitors, e.g.metformin or oligomycin, and pharmaceutical compositions comprisingsyrosingopine and mitochondrial inhibitors may be administeredespecially for cancer therapy in combination with radiotherapy,immunotherapy, surgical intervention, or a combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies or neo-adjuvant therapy incombination with surgery. Other possible treatments are therapy tomaintain the patient's status after tumor regression, or chemopreventivetherapy, for example in patients at risk.

The present invention relates furthermore to a method for the treatmentof cancer and of immunological disorders such as autoimmunity, whichcomprises administering a combination of syrosingopine and amitochondrial inhibitor, e.g. metformin or oligomycin, in a quantityeffective against said disease, to a warm-blooded animal requiring suchtreatment. The combination of syrosingopine and mitochondrialinhibitors, e.g. metformin or oligomycin, can be administered as such orespecially in the form of pharmaceutical compositions, prophylacticallyor therapeutically, preferably in an amount effective against the saiddiseases, to a warm-blooded animal, for example a human, requiring suchtreatment. In the case of an individual having a bodyweight of about 70kg the daily dose administered is from approximately 0.05 g toapproximately 3 g, preferably from approximately 0.25 g to approximately1.5 g, of a combination of the present invention.

The invention relates to the use of a combination of syrosingopine and amitochondrial inhibitor, e.g. metformin or oligomycin, and ofpharmaceutical compositions comprising syrosingopine and a mitochondrialinhibitor for the treatment of cancer, in particular for the treatmentof the particular cancers mentioned above. More specifically, theinvention relates to the use of a combination of syrosingopine and amitochondrial inhibitor and of pharmaceutical compositions comprisingsyrosingopine and a mitochondrial inhibitor for the treatment ofcarcinomas, sarcomas, leukemias, myelomas, lymphomas, and cancers of thenervous system. Furthermore, the invention relates to the use of acombination of syrosingopine and a mitochondrial inhibitor and ofpharmaceutical compositions comprising syrosingopine and a mitochondrialinhibitor for achieving immunosuppression in autoimmunity,transplantation medicine and in other cases where immunosuppression isdesirable, in particular in immunological diseases sensitive to blockadeof T cell proliferation, systemic autoimmune diseases, and localizedautoimmune diseases, as explained above. More specifically, theinvention relates to the use of a combination of syrosingopine and amitochondrial inhibitor, e.g. metformin or oligomycin, and ofpharmaceutical compositions comprising syrosingopine and a mitochondrialinhibitor for the treatment of autoimmune diseases, such as autoimmunediseases of the skin, nervous system, connective tissue, muscle, nervoussystem, blood forming system, bone and inner organs, in particularpsoriasis, multiple sclerosis, and anemias.

The preferred relative amount of syrosingopine and mitochondrialinhibitor, e.g. metformin or oligomycin, dose quantity and kind ofpharmaceutical composition, which are to be used in each case, depend onthe type of cancer or autoimmune disease, the severity and progress ofthe disease, and the particular condition of the patient to be treated,and has to be determined accordingly by the medical doctor responsiblefor the treatment.

The invention further relates to the use of a combination ofsyrosingopine and a mitochondrial inhibitor, e.g. metformin oroligomycin, for the preparation of a pharmaceutical composition for thetreatment of cancer or autoimmune disease, as explained above.

Especially, the invention provides a method for treatment of cancer orautoimmune disease, which comprises administering a combination ofsyrosingopine and a mitochondrial inhibitor, e.g. metformin oroligomycin, or of a pharmaceutical composition comprising syrosingopineand a mitochondrial inhibitor, in a quantity effective against saiddisease, to a warm-blooded animal requiring such treatment.

Rationale for the Use of a Combination of Syrosingopine and Metformin orOther Biguanides

A combination screen was performed, where cells were co-treated with theanti-diabetic agent metformin plus over a thousand drugs and drug-likecompounds. It was found that metformin and syrosingopine synergize tokill various cancer cells in vitro and in vivo. This effect is onlyobserved when the two drugs are combined, with minimal cytotoxicity foreach individual compound.

Mouse mast cell line 6.5 (Colombi et al., Oncogene 2011, 30:1551-65) wasused for a preliminary screen. 6.5 cells show many features of oncogenictransformation such as loss of growth-factor (IL3) dependence andsignaling pathway addiction. They are addicted to the PI3K-mTOR-Aktpathway as well as to the MAP kinase pathway as shown by inhibitorstudies. These cells display exquisite sensitivity to many clinicaldrugs. A drug co-screen using a commercial drug library (PrestwickChemical Library) on mouse 6.5 cells in the presence of metformin (2 mM)was performed to identify compounds that would act as a metforminco-drug to kill these cells synergistically.

The best hit identified was syrosingopine. FIG. 1A shows, that metforminalone at 5 mM triggers 20% inhibition of growth (left panel), whilesyrosingopine alone is virtually non-inhibitory (right panel, straightline). However, when increasing concentrations of syrosingopine arecombined with 5 mM metformin (right panel, dashed line), a dramaticinhibition is seen with 2.5 μM syrosingopine. Similar findings were madewith a series of human cancer lines, specifically the myeloma line OPM2(FIG. 1B), the myeloma line RPM18226 (FIG. 10), the T cell leukemia lineJurkat (FIG. 1D), the chronic myelogenous leukemia line K562 (FIG. 1E).Notably, in these cell lines the combined activity showed potentactivity at a concentration range where each compound used singly hadonly a marginal effect. In human fibrosarcoma line HT1080 (FIG. 1F) aswell as in two normal human fibroblast lines (FIG. 1G, H) no inhibitionof growth was observed, when cells were co-treated with both drugs. Thisis corroboration for a specific response rather than generalcytotoxicity, and the lack of activity on normal human fibroblastsindicates that the effect is tumor-specific. Table 1 summarizes datafrom these and additional lines tested, which together indicate, thatthe syrosingopine/metformin effect acts upon cancer cells of a widevariety of cancer histotypes, whereby the cell lines used have beenwidely used in preclinical cancer research and represent accepted andrepresentative models of organ-specific malignancy.

TABLE 1 Panel of human cancer cells tested for sensitivity to metforminand syrosingopine co-treatment. Human Cancer Cell Malignancy SensitivityOPM1 Multiple myeloma YES OPM2 Multiple myeloma YES RPMI8226 Multiplemyeloma YES A549 Lung cancer (NSCLC) YES H1299 Lung cancer (NSCLC) YESMDA-468 Breast cancer YES AN3CA Endometrial cancer YES Jurkat T cellleukemia YES K562 Chronic myeloid leukemia YES HL60 Promyelocyticleukemia YES KG1 Acute myeloblastic leukemia YES MOLT4 Acute lymphocyticleukemia YES HeLa Cervical cancer YES JUSO Melanoma YES PC3 Prostatecancer YES DU145 Prostate cancer YES LnCAP Prostate cancer YES LN229Glioblastoma YES HT1080 Fibrosarcoma NO MDA-231 Breast cancer NO U87Glioblastoma NO ME-59 Melanoma NO NA-8 Melanoma NO

A time course experiment of the combined treatment was performed on twosensitive human cancer cell lines, OPM2 (multiple myeloma) and A549(lung cancer). No out-growth was observed after 9-10 days of treatment(FIG. 2). To see whether the inhibition observed involved actualapoptotic cell death, drug-treated cells were analyzed for membranesurface expression of annexin V, a marker of apoptosis, and by propidiumiodide (PI) staining, revealing cell death. The number of apoptoticcells was increased with a corresponding decrease in the viable cellpopulation in OPM2 and RPMI8226 human multiple myeloma cells undergoingthe combined drug treatment, indicating apoptotic induction as themechanism of cell killing (FIG. 3). No apoptotic induction was observedwhen the compounds were used singly.

Syrosingopine of formula (4) is an artificial derivative of reserpine offormula (3), an anti-hypertensive agent, and shares therauwolfia-specific chemical backbone. Other rauwolfia-related compounds,such as reserpine, reserpinnic acid, rescinnnamine, yohimbinic acid,corynanthine HCl, ajmalicine, yohimbine HCl, and rauwolscine HCl do notshow significant interaction with metformin of formula (1) in cancertest cell lines. To confirm the specificity for syrosingopine, reserpinewas tested over a range of concentrations in 6.5 and OPM2 cells. Nointeraction of reserpine with metformin was observed (FIG. 4),suggesting that syrosingopine is acting in a novel fashion compared toother rauwolfia derivatives.

To test if the interaction also holds in an in vivo context, 6.5 cellswere injected into immunocompatible DBA mice. Treatment was begun whenthe tumors reached 100 mm² in size. Mice were injectedintra-peritoneally daily with syrosingopine (2 mg/kg body weight),metformin (250 mg/kg body weight) or both for 15 days and sacrificed fortumor dissection and measurement. As seen in FIG. 5A, the dual treatmentsuccessfully arrested tumor growth. There was no significant differencein body weight between the treatment groups (FIG. 5B) or any signs ofovert toxicity.

Metformin is a biguanide similar to phenformin (formula 2), anantidiabetic compound which is rarely in clinical use today due to sideeffects (lactic acidosis). It was tested whether phenformin alsosynergizes with syrosingopine. As shown in FIG. 6, left panel,phenformin treatment alone leads to no growth inhibition atconcentrations up to 10 μM. When phenformin at this concentration,however, is combined with increasing concentrations of syrosingopine(FIG. 6, right panel), a strong synergistic effect is seen. In fact, adose dependent synergistic effect is observed at all concentrations ofphenformin tested.

The triggering of an immune response by antigenic stimulation involvesearly proliferation of T cells. This T cell stimulation can be mimickedby polyclonal stimulators such as lectins, for examplephytohaemagglutinin (PHA). The combination of metformin andsyrosingopine also inhibits PHA-stimulated T cell proliferation.Leukocytes isolated from the peripheral blood of normal human blooddonors were tested, and proliferation measured on day 3 after incubationwith metformin, syrosingopine, or both. As shown in FIG. 7, right panel,PHA-stimulated proliferation is very sensitive to inhibition whensyrosingopine is combined with metformin (4 mM) (dashed line), butconsiderably less so in absence of metformin (solid line). The leftpanel of FIG. 7 shows peripheral blood leukocytes cultured for 3 days inthe absence of PHA, where cells hardly proliferate but survive. Thissurvival is not affected by the presence of increasing concentrations ofsyrosingopine in presence of 4 mM metformin (dashed line).

Rationale for the Use of a Combination of Syrosingopine and aMitochondrial Inhibitor Other Than Metformin

The fact that metformin blocks complex I of the ETC raised the questionwhether other inhibitors of the ETC or other mitochondriotoxic agentsmay similarly co-operate with syrosingopine in cell killing. A series ofagents well known to target mitochondria was tested, and concentrationsof these agents were used which are not or only minimally toxic bythemselves. The agents and the concentrations used are shown in FIGS. 8and 9. When tested in collaboration with increasing amounts ofsyrosingopine, synergistic killing with the ETC complex I inhibitorsrotenone (FIG. 8A), piericidin A (FIG. 8B), epiberberine (FIG. 8C) wasdetected. Synergistic killing was likewise detected with the complex IIinhibitors TTFA (FIG. 8D) and sodium malonate (FIG. 8E), as well as withthe complex III inhibitor antimycin A (FIG. 8F). Two complex IVinhibitors, namely KCN (FIG. 8G) and sodium azide (FIG. 8H) as well asthe complex V inhibitor oligomycin (FIG. 8I) also induced synergisticcell killing with syrosingopine. These data establish that syrosingopineinduces cell killing when applied together with mitochondrialinhibitors, in this case with ETC inhibitors. FCCP is an agent whichuncouples the ETC chain from ATP synthesis. Importantly, this agent alsoinduced synergistic killing with syrosingopine (FIG. 8J). As severalcomponents of the ETC are encoded by the mitochondrial genome, one wouldexpect that agents which damage the mitochondrial genome would similarlysynergize with syrosingopine. The anti-HIV compound stavudine, a reversetranscriptase inhibitor with well described detrimental side effects onmitochondrial genome replication, was tested. As shown in FIG. 8K,stavudine indeed induced synergistic killing with syrosingopine. Ofnote, the HIV reverse transcriptase inhibitor 3TC (also known aslamivudine and lacking anti-mitochondrial toxicity) did not synergizewith syrosingopine. The results of these experiments lead to theconclusion that ETC inhibitors in general, but also anti-mitochondrialagents which compromise ETC function indirectly, synergize withsyrosingopine and are anti-cancer agents and immunosuppressive agentswhen used at non-toxic concentrations but in combination withsyrosingopine.

For practical reasons, the salts sodium malonate, potassium cyanide andsodium azide, although shown to synergize with syrosingopine in celltests, are not considered combination partners for syrosingopine asanti-cancer and immunosuppressive agents. An interesting combinationpartner for syrosingopine, however, is for example oligomycin, as evenat a concentration below 1 nM this compound kills human cancer lines incombination with syrosingopine (FIG. 10), being without toxic effect inthe absence of syrosingopine (FIG. 9).

In Vitro Test Indicating Syrosingopine Sensitivity

In view of the foreseen application of syrosingopine to cancer therapy,it is important to have an in vitro test predicting sensitivity of acancer cell. Since there are good reasons that the functional target ismitochondrial (as with metformin), the effect of syrosingopine on themembrane potential of the inner mitochondrial membrane was tested. Themembrane is more positively charged outside (i.e. in the inter-membranespace) than inside (in the mitochondrial matrix) as mitochondrialrespiration involves extrusion of protons from the mitochondrial matrixthrough the inner membrane into the inter-membrane space. The matrix,being more negatively charged, accumulates positively charged lipophilicmolecules such as TMRM (tetramethylrhodamine methyl ester). As TMRM isfluorescent, it functions as a potentiometric dye and lends itself toeasy assessment of mitochondrial membrane potential. In FIG. 11 (panels3-6) it is shown that increasing amounts of syrosingopine lead to anincrease of the fluorescent TMRM signal (shift to the right) indicatingincreased positive membrane polarity. The fluorescence peak shifts from403 (panel 1) to 955 (panel 9) arbitrary units. Use of FCCP, anuncoupling agent used for control (panel 2), leads to the expectedcollapse of the membrane potential (as shown by the left-shift of thefluorescence peak down to 42 arbitrary units). While the perturbation ofthis critical membrane function by syrosingopine is likely to contributeto its synergistic activity with metformin, a known weak mitochondrialinhibitor, the discovery of a mitochondrial effect of syrosingopineallows to perform a straightforward test for syrosingopine-sensitivityof cancer cells.

Based on these results, the invention further relates to a method forthe determination whether a cancerous cell is responsive tosyrosingopine treatment comprising the steps of

(a) preparation of single cell suspension and culturing the cancerouscell in a suitable media,

(b) incubating the cancerous cell with syrosingopine,

(c) incubating the cancerous cell of step (b) with a positively chargedfluorescent dye,

(d) measuring the excitation fluorescence intensity, and

(e) comparing the measured fluorescence intensity of step (d) with themeasured fluorescence intensity of the cancerous cell incubated with thepositively charged fluorescent dye alone,

and wherein a relative increase of fluorescence intensity of cancerouscells pre-incubated with syrosingopine indicates syrosingopine treatmentresponsiveness.

For practical purposes the cancerous cell is a cell isolated from apotential patient to be treated with a combination of syrosingopine anda mitochondrial inhibitor. Suitable media for culturing such cancerouscells are well known in the art, and include, for example Iscovesmodified Dulbecco medium (IMDM) or RPMI 1640 medium. Prior to testing, asingle cell suspension from the ex vivo tumor material has to beprepared. Again, suitable standardized commercial methodologies are athand, where physical disruption and enzymatic digestion steps arecombined (see for example the method by MiltenyiBiotec(http://www.miltenyibiotec.com/downloads/6760/6764/30501/PDF1.pdf) or byInvitrogen(http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.18492.File.dat/DissociationCells Y14477 Dissociation.pdf)). For testing, it is advisable topreincubate the cancerous cell with different concentrations ofsyrosingopine, e.g. 0.1 μM and 10 μM, for 2 to 8 h. A suitablepositively charged fluorescent dye is TMRM (tetramethylrhodamine methylester perchlorate). Other positively charged fluorescent dyes consideredare the rhodamines TMRE (tetramethylrhodamine ethyl ester perchlorate),Rhodamine 123 (rhodamine methyl ester chloride), Rhodamine B(tetraethylrhodamine hydrochloride), MitoTracker Red CMXRos® (CASdesignation1H,5H,11H,15H-xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium,9[4-(chloromethyl)phenyl]-2,3,6,7,12,13,16,17-octahydro-, chloride), andthe carbocyanines JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanineiodide) and DiOC₆(3) (3,3′-dihexylbenzoxazolocarbocyanine iodide).

Staining with the preferred fluorescent dye TMRM (tetramethylrhodaminemethyl ester) is preferably done according to standard methods, such asindicated by the supplier Serotec. Fluorescence is measured at 575 nmafter excitation at 488 nm, preferably in a standard commerciallyavailable flow cytometer.

If the cancerous cell shows responsiveness to syrosingopine, thecorresponding patient will probably be effectively treated bycombinations of syrosingopine and a mitochondrial inhibitor. If thecancerous cell is not responsive to syrosingopine in the correspondingfluorescence test with TMRM, chances are low that the patient can beeffectively treated with combinations of syrosingopine and amitochondrial inhibitor.

EXAMPLES Cell culture

Mouse mast cell line 6.5 (Colombi et al., Oncogene 2011, 30:1551-65) wascultured in IMDM, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100μg/ml streptomycin and 50 μM 2-mercaptoethanol, exogenous IL-3 was addedas 1% conditioned medium from X63 murine IL3 secreting cells. Humanleukemia cell lines Jurkat, K562, OPM1, OPM2, RPMI8226 were grown inRPMI-1640 medium supplemented with 10% FCS, 2 mM L-glutamine.Recombinant human IL-6 (Biomol) was added at 10 ng/ml to the media forOPM2 and RPMI8226. Other human cancer lines MDA-468, MDA-231, A549,H1299, AN3CA, JUSO, HT1080, ME-59, HL60, KG1, MOLT4, HeLa, PC3, DU145,LnCAP.

LN229, U87 and NA-8 were grown in Iscove's medium containing 10% FCS, 2mM L-glutamine and 50 μM 2-mercaptoethanol. All cells were grown at 37°C. in 5% CO₂.

Reagents

Metformin and phenformin (Sigma) were prepared as 1 M stocks in PBS andkept at 4° C., syrosingopine (Extrasynthese) and reserpine (Sigma) wereprepared as 5 mM stock in DMSO and stored at −20° C. Stocks ofpieridicin A, epiberberine, TTFA, antimycin A, oligomycin (Sigma) andFCCP were prepared in DMSO; sodium malonate, KCN and NaN₃ were dissolvedin sterile, distilled water, and rotenone and stavudine stock solutionswere made in absolute ethanol.

Mouse Mast Cell Line 6.5

The cell line 6.5 has recently been described (Colombi et al., Oncogene2011, 30:1551-65). These cells were generated by treating 15V4 mastcells (Nair et al., Oncogene 1992, 7:1963-72) with mutagen ICR191, whichled to loss of the Pten tumor suppressor gene.

This loss abrogated the IL-3 dependence of the cells and generatedgrowth autonomous cells which formed tumors in syngeneic mice.

Inhibitor Studies of Mouse Mast Cell Line 6.5

Mouse mast cell line 6.5 cells are addicted to the PI3K-mTOR-Akt pathwayas well as to the MAP kinase pathway as shown to their sensitive tonanomolar concentrations of the mTOR-inhibitor rapamycin (Colombi etal., Oncogene 2011, 30:1551-65) or the MEK inhibitor U0126.

Cell Proliferation Assay

Cells were seeded at appropriate density (5,000-15,000 cells per welldepending on cell type, 150 μl medium per well) in flat-bottomed 96-wellplates and compounds added at the desired concentrations. After 3 days,proliferation was assayed by adding 0.1 vol. AlamarBlue (Invitrogen),and fluorescence was read at 535/595 nm excitation/emission after 4-6hours of color development. Readings were normalized to non-treatedcontrol cells and growth expressed as percentage of control growth.

Apoptosis Assay

Apoptosis was determined by Annexin V-FITC (Invitrogen) and propidiumiodide counter-staining, and cells were analysed by FACS to distinguishbetween viable/early apoptotic/late apoptotic cell sub-populations.Cells were seeded at a density of 100,000 cells/5 ml medium withaddition of compounds as indicated. 500 μl of culture was harvestedafter 3 days and the cells were washed and stained with PI/Annexin Vbefore FACs analysis.

Mouse Syngeneic Tumor Model

4×10⁵ 6.5 cells were injected in 150 μl of PBS into the flanks ofimmuno-compatible DBA mice. Tumor progression was monitored andtreatment was started when tumor area reached 100 mm² in size. Mice wereinjected intra-peritoneally daily with metformin (250 mg/kg bodyweight), syrosingopine (2 mg/kg body weight) or combined for 15 daysbefore being sacrificed for tumor tissue measurements.

A Combination of Metformin and Syrosingopine Kills Mouse Mast Cells 6.5

A drug co-screen was performed using a commercial drug library(Prestwick Chemical Library) on mouse 6.5 cells in the presence ofmetformin (2 mM) to identify compounds that would act as a metforminco-drug to kill these cells synergistically.

Effect of Metformin and sysrosingopine on Phytohaemagglutinin-StimulatedT Cell Proliferation

Normal human buffy coat cells were obtained from a local blood donationcenter in accordance with the ethical guidelines. 80 ml of blood wasdiluted with 200 ml of Iscove's medium and overlayed on a Ficollgradient. The gradient was centrifuged at 1400×g for 5 minutes toseparate the leukocyte fraction from erythrocytes. Cells were plated oncoated tissue culture dishes for 1 hour to remove adherent cells, andthe remaining suspension cells were collected by centrifugation andcounted with a haemocytometer. 50,000 cells were seeded per well in96-well dishes and compounds were added according to the varioustreatment regimes to a final medium volume of 150 μl. For stimulation,phytohemagglutinin was added to a final concentration of 10 μg/ml. Atday 3, proliferation was determined by AlamarBlue assay.

Measurement of Membrane Potential of Mitochondrial Membrane by TMRMFluorescence Assay

Murine 6.5 cells were treated for 20 min with the potentiometricfluorescent dye TMRM (tetramethylrhodamine methyl ester, AbD Serotec,100 nM) and the fluorescence intensity was measured at 575 nm emissionafter excitation at 488 nm using a flow cytometer (CellLabQuanta,Beckman Coulter). This dye accumulates in the mitochondrial matrix whenthe inner membrane potential is more positive outside due to theextrusion of protons. For control, cells were pretreated for 1 h withthe uncoupling agent FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) which dissipates the membranepotential and abolishes fluorescence intensity. The effect ofsyrosingopine was assessed by pre-incubating cells with increasingconcentrations of syrosingopine for 4 h as indicated, followed by TMRMstaining.

1-19. (canceled)
 20. A method for treatment of cancer or autoimmunedisease, which comprises administering a combination of syrosingopineand a mitochondrial inhibitor, or of a pharmaceutical compositioncomprising syrosingopine and a mitochondrial inhibitor, in a quantityeffective against said disease, to a warm-blooded animal requiring suchtreatment.
 21. The method of claim 20 wherein the mitochondrialinhibitor is metformin.
 22. The method of claim 20 wherein themitochondrial inhibitor is oligomycin.
 23. The method of claim 20wherein the cancer is selected from the group consisting of carcinoma,sarcoma, leukemia, myeloma, lymphoma, and cancers of the nervous system.24. The method of claim 20 for use in immunosuppressive treatment of anautoimmune disease.
 25. The method of claim 20 wherein the autoimmunedisease is selected from the group consisting of autoimmune diseases ofthe skin, nervous system, connective tissue, muscle, nervous system,blood forming system, bone and inner organs.
 26. The method of claim 20wherein the combination of syrosingopine and the mitochondrial inhibitoris part of the same pharmaceutical composition.
 27. The method of claim20 wherein the combination of syrosingopine and the mitochondrialinhibitor is in different pharmaceutical compositions.